Solid-solid separation process for long chain dicarbozylic acids

ABSTRACT

The present disclosure provides method for isolating a long chain dicarboxylic acid such as a substantially pure or pure long chain dicarboxylic acid from a fermentation broth containing microbial cells.

PRIORITY CLAIM

This application is a Continuation of co-pending prior application U.S.Ser. No. 16/339,756, filed on Apr. 5, 2019, entitled “Solid-SolidSeparation Process for Long Chain Dicarboxylic Acids”, which is thenational stage entry of PCT/CN2016/089615, filed on Jul. 11, 2016, theentire disclosure of which is herein incorporated by reference.

BACKGROUND OF THE INVENTION

Long chain carboxylic acids are useful for the production of nylons andother polyamides, resins, polyesters, fragrances or perfumes, adhesives,powder coatings, corrosion inhibitors, lubricants, plasticizers, andpharmaceutical products. These carboxylic acids can be generated on anindustrial-scale from long-chain n-alkanes and/or fatty acids viafermentation of microbial cells.

The standard method used to separate long chain dicarboxylic acids froma fermentation broth can include: (1) adjusting the pH of the broth toabout 8 to about 10 by adding a base to the broth, (2) heating the brothand applying membrane filtration to remove cells and obtain a clearbroth containing dibasic acid salts; (3) adding an acid such as sulfuricacid (H₂SO₄) to precipitate the dibasic acid; and (4) filtration anddrying the precipitated dibasic acid to recover the long chaindicarboxylic acid product.

For instance, Chinese Patent No. CN1053470C describes a method thatincludes adding an alkali to a fermentation broth containing brassylicacid (DC13) to adjust the pH value at the range of 10-12. The broth isthen heated to 85-90° C. and demulsified. In some cases, the upper layerof the residual oil is recycled. The middle layer is collected as aclear broth. The cells layer can be re-treated to obtain a second clearbroth or be sent directly for centrifugation or filtration. The clearbroth is then decolorized using activated carbon for 30 min at 85-90° C.Thereafter the activated carbon can be removed. The decolored solutionis heated to 60-70° C. and either HCl or H₂SO₄ is added into thedecolored solution to adjust the pH value range to 4-5 for acidificationand crystallization to obtain the long chain diacid product.

The pH of the starting fermentation broth is usually about pH 7-8. Inthis method, a large amount of alkali is used to demulsify the broth andto remove the alkane and cells. For example, the pH of the broth istypically raised to about pH 10-12. After the acidification step, excessalkali is also used to neutralize the corresponding excess acid. As aresult, excess salts are produced and discharged into the waste water.

Chinese Patent No. CN97121846.3 describes a method that includesdemulsifying a fermentation broth by adding alkali, and then removingthe microbial cells by filtration. The broth is then acidified to obtaina crude dicarboxylic acid. Alkali and water are added to the crude acidto dissolve the salt, and recover the salt by a salting-out method. Therecovered salt is then dissolved in water and filtered to removeimpurities. Next, the filtrate is then acidified to obtain the desiredlong chain dicarboxylic acid. A key disadvantage of this method is theconsumption of large amounts of acids and alkali.

In the separation method described in Chinese Patent ApplicationPublication No. CN103570525A, the residual alkane and microbial cellsare separated from the clear broth phase by centrifugation. An alkalisolution (about 4 mg/L-400 mg/L alkali solution) is added to theseparated cell phase, and then the cells are lysed and separated bymembrane filtration. The membrane filtrate is combined with the clearbroth phase. Alkali is then added to the mixture to adjust the pH toabout 8.0-10.5. This treated mixture then undergoes decoloration,separation and acidification to produce the desired long chaindicarboxylic acid.

The conventional process for extracting dicarboxylic acids from afermentation broth is (1) to dissolve the acids using an alkali, (2) toremove the cells by filter or membrane filtration or centrifugation, and(3) to acidify the dicarboxylic acid salt. Because of the high viscosityof fermentation broths, it can be difficult to separate cells from thebroth by using standard filtering equipment. To solve such problems, amicrofiltration membrane can be used. However, membrane filtrationequipment is extremely costly and requires a large amount of power tooperate. Also, the membrane units typically last for only 2-3 yearsbefore expensive servicing is required. In some cases, excess alkalimust be added into the fermentation broth to increase the pH value atthe range of 8.5-11.0 for full demulsification and proper extraction. Toproduce the desired dicarboxylic acid, the excess alkali must beneutralized by acidification which in turn can produce a large amount ofsalt in the wastewater. Also the precipitated dicarboxylic acids must beseparated from the acidified solution by appropriate equipment.Repeating filtering and separating can have adverse effects on theproduct yield.

For instance, such a demulsifying method is described Chinese Patent No.CN104693018A. The fermentation broth of long chain diacid is directlyacidified, and both the precipitated dicarboxylic acid and microbialcells are separated from the broth and collected together. They are thendried. Eventually, the cells are removed in subsequent purificationprocesses that use an organic solution. A key problem with this methodis the presence of organic impurities which can be insoluble in anorganic solvent. As such, the method leads to a risk of low productpurity and high product loss.

Isolating substantially pure or pure dicarboxylic acids fromfermentation broths remains a challenge.

BRIEF SUMMARY OF THE INVENTION

In one aspect, described herein is a method for isolating a long chaindicarboxylic acid from a fermentation broth containing microbial cells.The method includes (a) adding an acid to the fermentation broth toproduce a demulsified fermentation broth having a pH of 6 or less; (b)centrifuging the demulsified fermentation broth to generate a firstphase comprising the long chain dicarboxylic acid and a second phasecomprising the microbial cells; and (c) isolating the long chaindicarboxylic acid from the first phase to recover an isolated long chaindicarboxylic acid.

The method can further comprise heating the fermentation broth to atemperature from about 90° C. to about 105° C. prior to step (a).Alternatively, the method can further comprise heating the demulsifiedfermentation broth to a temperature from about 90° C. to about 105° C.prior to step (b). The method can also comprise cooling the demulsifiedfermentation broth to a temperature from about 30° C. to about 80° C.after heating (e.g., heating prior to step (a) and heating prior to step(b)).

In some embodiments, the first phase is substantially free of themicrobial cells. The second phase can be substantially free of the longchain dicarboxylic acid. In some instances, the fermentation broth is asterilized fermentation broth.

In some embodiments, the acid is selected from the group consisting ofsulfuric acid, hydrochloric acid, nitric acid, acetic acid, phosphoricacid, trifluoromethane sulfonic acid, and any combination thereof. Insome instances, the acid is sulfuric acid. In some cases, thedemulsified fermentation broth has a pH of 2-5. In other cases, the pHis from pH 2.5-4. In some embodiments, the demulsified fermentationbroth has a viscosity of about 5 centipoise (cp)-about 200 cp.

Centrifuging can comprise sequential centrifugation. In some instances,centrifuging is performed using a scanter centrifuge. The scantercentrifuge can be selected from the group consisting of a hydrauliccyclone centrifuge, a hydrocyclone centrifuge, a horizontal spiralcentrifuge, and any combination thereof.

In some embodiments, the method further comprises filtering the isolatedlong chain dicarboxylic acid. In some instances, the method furthercomprises drying the isolated long chain dicarboxylic acid.

The isolated long chain dicarboxylic acid can have a purity of at least95% or higher. Alternatively, the long chain dicarboxylic acid has apurity of at least 97% or higher. In some cases, the long chaindicarboxylic acid is a saturated or unsaturated straight chaindicarboxylic acid having 9 to 18 carbon atoms, with a carboxyl group attwo ends of the chain. In other cases, the long chain dicarboxylic acidis a saturated or unsaturated straight chain dicarboxylic acid having 11to 14 carbon atoms, with a carboxyl group at each of the two ends of thechain. In some embodiments, the long chain dicarboxylic acid is selectedfrom the group consisting of a nonanedioic acid, decanedioic acid,undecanedioic acid, dodecanedioic acid, tridecanedioic acid,tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid,heptadecanedioic acid, octadecanedioic acid, 9-ene-octadecanedioic acid,and any combination thereof. In some instances, the long chaindicarboxylic acid is selected from the group consisting of anundecanedioic acid, dodecanedioic acid, tridecanedioic acid,tetradecanedioic acid, and any combination thereof.

In another aspect, described herein is a method for isolating a longchain dicarboxylic acid from a fermentation broth containing microbialcells. The method comprises: (a) adding sulfuric acid to thefermentation broth to produce a demulsified fermentation broth having apH of 2.5-4; (b) heating the demulsified fermentation broth to atemperature from 90° C. to 105° C.; (c) centrifuging the demulsifiedfermentation broth to generate a first phase comprising the long chaindicarboxylic acid and a second phase comprising the microbial cells,wherein the first phase is substantially free of the microbial cells andthe second phase is substantially free of the long chain dicarboxylicacid; and (d) isolating the long chain dicarboxylic acid from the firstphase to recover an isolated long chain dicarboxylic acid.

In yet another aspect, provided herein is a method for isolating a longchain dicarboxylic acid from a fermentation broth containing microbialcells. The method comprises: (a) heating the demulsified fermentationbroth to a temperature from 90° C. to 105° C.; (b) adding sulfuric acidto the fermentation broth to produce a demulsified fermentation brothhaving a pH of 2.5-4; (c) centrifuging the demulsified fermentationbroth to generate a first phase comprising the long chain dicarboxylicacid and a second phase comprising the microbial cells, wherein thefirst phase is substantially free of the microbial cells and the secondphase is substantially free of the long chain dicarboxylic acid; and (d)isolating the long chain dicarboxylic acid from the first phase torecover an isolated long chain dicarboxylic acid.

In some embodiments, the method for isolating a long chain dicarboxylicacid from a fermentation broth containing microbial cells comprises: (a)adding sulfuric acid to the fermentation broth to produce a demulsifiedfermentation broth having a pH of 2.5-4; (b) heating the demulsifiedfermentation broth to a temperature from 90° C. to 105° C.; (c) coolingthe demulsified fermentation broth down to about 80° C. to about 30° C.;(d) centrifuging the demulsified fermentation broth to generate a firstphase comprising the long chain dicarboxylic acid and a second phasecomprising the microbial cells, wherein the first phase is substantiallyfree of the microbial cells and the second phase is substantially freeof the long chain dicarboxylic acid; and (e) isolating the long chaindicarboxylic acid from the first phase to recover an isolated long chaindicarboxylic acid.

In other embodiments, the method for isolating a long chain dicarboxylicacid from a fermentation broth containing microbial cells comprises: (a)heating the demulsified fermentation broth to a temperature from 90° C.to 105° C.; (b) cooling the demulsified fermentation broth down to about80° C. to about 30° C.; (c) adding sulfuric acid to the fermentationbroth to produce a demulsified fermentation broth having a pH of 2.5-4;(d) centrifuging the demulsified fermentation broth to generate a firstphase comprising the long chain dicarboxylic acid and a second phasecomprising the microbial cells, wherein the first phase is substantiallyfree of the microbial cells and the second phase is substantially freeof the long chain dicarboxylic acid; and (e) isolating the long chaindicarboxylic acid from the first phase to recover an isolated long chaindicarboxylic acid.

In yet another aspect, described herein is a substantially pure longchain dicarboxylic acid isolated according to any one of the methodsprovided herein.

In some embodiments, the substantially pure long chain dicarboxylic acidhas a purity of at least 95% or more. In other embodiments, thesubstantially pure long chain dicarboxylic acid has a purity of at least97% or more. The substantially pure long chain dicarboxylic acid can bea saturated or unsaturated straight chain dicarboxylic acid having 9 to18 carbon atoms, with a carboxyl group at each of the two ends of thechain. Alternatively, the substantially pure long chain dicarboxylicacid can be a saturated or unsaturated straight chain dicarboxylic acidhaving 11 to 14 carbon atoms, with a carboxyl group at each of the twoends of the chain. In certain cases, the substantially pure long chaindicarboxylic acid is selected from the group consisting of anundecanedioic acid, dodecanedioic acid, tridecanedioic acid,tetradecanedioic acid, and any combination thereof.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The invention provides an effective method for solid-solid separation toisolate a long chain dicarboxylic acid from a fermentation broth. Thisinvention provides a more efficient isolation or extraction process thatincludes heating the broth and directly adding an acid to precipitatethe dibasic acid. At this step, the broth emulsion is broken, andprecipitation of the dicarboxylic acid increases such thatcentrifugation can be used to separate cells from the dicarboxylic acidparticles. The method is simple with fewer steps and requires lessmonetary investment. In addition, the consumption of acids and bases aregreatly reduced compared to standard method. As such, fewer salts(typically Na₂SO₄) are found in the waste water, which reduces wastewater treatment costs and minimizes the potential of environmentaldamage.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forinstance, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the agent” includes reference to one or more agents knownto those skilled in the art, and so forth.

The terms “about” and “approximately” shall generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Typical, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values. Alternatively, and particularly inbiological systems, the terms “about” and “approximately” may meanvalues that are within an order of magnitude, preferably within 5-foldand more preferably within 2-fold of a given value. Numerical quantitiesgiven herein are approximate unless stated otherwise, meaning that theterm “about” or “approximately” can be inferred when not expresslystated.

The term “long chain dicarboxylic acid” refers to an α,ω-dicarboxylicacid. Non-limiting examples of long chain carboxylic acids includenonanedioic acids (azelaic acids), decanedioic acids, undecanedioicacids, dodecanedioic acids, tridecanedioicaicds (brassylic acids),tetradecanedioic acids, pentadecanedioic acids, hexadecanedioic acids,heptadecanedioic acids, octadecanedioic acids, variants thereof, andderivatives thereof.

The term “mixture” refers to an aqueous liquid composition suitable forextraction by means described herein allowing the presence of somedicarboxylic acid, preferably more than 80%, more preferably than 95% byweight.

The term “fermentation broth”, “fermentation stream”, or “fermentationliquor” refers to an aqueous stream comprising one or more types ofcarboxylic acids that have been synthesized by a microbe(microorganism). Microorganisms that may be employed in the fermentationinclude wild-type or recombinant Escherichia, Zymomonas, Candida,Pichia, Streptomyces, Bacillus, Lactobacillus and Clostridium. In someembodiments, a fermentation broth includes one type of carboxylic acid.In other embodiments, a fermentation broth includes at least two typesof carboxylic acids.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. For example, recombinant cells express genes that are notfound within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under-expressed,or not expressed at all.

The term “sterilized fermentation broth” refers to a fermentation broththat has been treated by heating to kill or inactivate the microbialcells present in the broth.

The term “demulsified”, in the context of a fermentation broth, refersto a mixture that has been separated into some or all of its constituentcomponents such that it is unable to reform the original mixture withoutaid.

The term “substantially free of” refers to a composition that is about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% or more free of undesired components.

The term “substantially pure” refers to a composition that is about 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%or more pure.

The term “sequential centrifugation” refers to performing two or morecentrifugations in succession without performing an intervening stepother than moving the mixture or composition between centrifuges.

The term “isolated” or “separated” refers to a chemical compound,molecule, cell and the like that is separated from other components withwhich it is associated in a natural or unnatural state. The term doesnot mean that the preparation is technically pure (homogeneous), butrather that it is sufficiently pure to provide the chemical compound,molecule, cell and the like in a form in which it can be used for theintended purpose. In certain embodiments, the isolated compositioncomprising the dicarboxylic acid is at least about 95%, 96%, 97%, 98%,or 99% or more pure.

III. DETAILED DESCRIPTIONS OF EMBODIMENTS

Provided herein is a method for isolating a long chain dicarboxylic acidfrom a fermentation broth containing microbial cells. The methodincludes (a) adding an acid to the fermentation broth to produce ademulsified fermentation broth having a pH of 6 or less; (b)centrifuging the demulsified fermentation broth to generate a firstphase, e.g., a heavy phase, comprising the long chain dicarboxylic acidand a second phase, e.g., a light phase, comprising the microbial cells;and (c) isolating the long chain dicarboxylic acid from the first phaseto recover an isolated long chain dicarboxylic acid.

In some embodiments, the fermentation broth includes microbial cells. Insome cases, the fermentation broth is a diluted fermentation broth. Thefermentation broth or diluted fermentation broth can include at least0.5% microbial cells (dry weight), e.g., 0.5%, 0.6%, 0.7%, 0.8%, 0.9%,1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or more microbialcells (dry weight). In some instances, the fermentation broth includesabout 0.5% to about 10% microbial cells, e.g., about 0.5% to about 10%,about 0.5% to about 9%, about 0.5% to about 8%, about 0.5% to about 7%,about 0.5% to about 6%, about 0.5% to about 5%, about 0.5% to about 4%,about 0.5% to about 3%, about 0.5% to about 2%, about 0.5% to about 1%,about 1% to about 10%, about 1% to about 9%, about 1% to about 8%, about1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% toabout 4%, about 1% to about 3%, about 1% to about 2%, about 0.5% toabout 1%, about 1% to about 2%, about 2% to about 3%, about 3% to about4%, about 4% to about 5%, about 5% to about 6%, about 6% to about 7%,about 7% to about 8%, about 8% to about 9%, about 9% to about 10%, ormore microbial cells. In some embodiments, the fermentation brothcontains 10% microbial cells. Non-limiting examples of microbial cellsinclude Escherichia, Zymomonas, Candida, Pichia, Streptomyces, Bacillus,Lactobacillus and Clostridium. The microbial cells can be natural(wild-type) or recombinant. In some instances, the microbial cells ofthe fermentation broth are not removed via filtration or centrifugation.

In some instances, the concentration of all dicarboxylic acids in thefermentation broth is at least about 85% (dry weight), e.g., about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, or more (dry weight). The totalconcentration of dicarboxylic acids in the fermentation broth can be atleast about 90% (dry weight).

In some embodiments, the first phase, e.g., the heavy phase, issubstantially free of the microbial cells. The first phase or heavyphase can include less than 0.5% (w/w) microbial cells, e.g., 0.4%(w/w), 0.3% (w/w), 0.2% (w/w), 0.1% (w/w) or no microbial cells. Thefirst phase or heavy phase can contain about 0% to about 0.4% (w/w),e.g., about 0% (w/w) to about 0.4% (w/w), about 0% to about 0.3% (w/w),about 0% (w/w) to about 0.2% (w/w), about 0% (w/w) to about 0.1% (w/w),about 0.1% (w/w) to about 0.4% (w/w), about 0.1% (w/w) to about 0.3%(w/w), about 0.1% (w/w) to about 0.2% (w/w), about 0.2% (w/w) to about0.4% (w/w) or about 0.2% (w/w) to about 0.3% (w/w) microbial cells. Insome cases, the first phase or heavy phase is free of microbial cells.In some instances, at least 80% (w/w), e.g., 80% (w/w), 81% (w/w), 82%(w/w), 83% (w/w), 84% (w/w), 85% (w/w), 86% (w/w), 87% (w/w), 88% (w/w),89% (w/w), 90% (w/w), 91% (w/w), 92% (w/w), 93% (w/w), 94% (w/w), 95%(w/w), 96% (w/w), 97% (w/w), 98% (w/w), 99% (w/w), or more of themicrobial cells of the fermentation broth or the demulsifiedfermentation broth are present in the second phase or light phase.

The method described herein can remove at least about 80%, e.g., about80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%,about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%of the microbial cells in the fermentation broth. In some cases, about80%-about 90%, about 80%-about 99%, about 85%-about 90%, about 80%-about95%, about 85%-about 95%, about 85%-about 99%, about 90%-about 95%, orabout 95%-about 99% of the cells can be removed from the fermentationbroth.

In some embodiments, the first phase, e.g., heavy phase, contains about10% (w/w) to about 80% (w/w) long chain dicarboxylic acid, e.g., about10% (w/w) to about 80% (w/w), about 20% (w/w) to about 80% (w/w), about30% (w/w) to about 80% (w/w), about 40% (w/w) to about 80% (w/w), about50% (w/w) to about 80% (w/w), about 60% (w/w) to about 80% (w/w), about10% (w/w) to about 60% (w/w), about 10% (w/w) to about 40% (w/w), about40% (w/w) to about 80% (w/w), about 20% (w/w) to about 60% (w/w), about40% (w/w) to about 60% (w/w), about 10% (w/w), about 20% (w/w), about30% (w/w), about 40% (w/w), about 50% (w/w), about 60% (w/w), about 70%(w/w), or about 80% (w/w) long chain dicarboxylic acid. In someinstances, at least 80% (w/w), e.g., 80% (w/w), 81% (w/w), 82% (w/w),83% (w/w), 84% (w/w), 85% (w/w), 86% (w/w), 87% (w/w), 88% (w/w), 89%(w/w), 90% (w/w), 91% (w/w), 92% (w/w), 93% (w/w), 94% (w/w), 95% (w/w),96% (w/w), 97% (w/w), 98% (w/w), 99% (w/w) or more of the long chaindicarboxylic acid in the fermentation broth or the demulsifiedfermentation broth is present in the first phase or heavy phase. Theconcentration or amount of long chain dicarboxylic acid can bedetermined by, for example, gas chromatography.

In some embodiments, the second phase, e.g., the light phase, issubstantially free of long chain dicarboxylic acid. This phase caninclude less than 10% (w/w) long chain dicarboxylic acid, e.g., 9.8%(w/w), 9.5% (w/w), 9.0% (w/w), 8.5% (w/w), 8.0% (w/w), 7.5% (w/w), 7.0%(w/w), 6.5% (w/w), 6.0% (w/w), 5.5% (w/w), 5.0% (w/w), 4.5%, 4.0% (w/w),3.5% (w/w), 3.0% (w/w), 2.5% (w/w), 2.0% (w/w), 1.5% (w/w), 1.0% (w/w),0.5% (w/w), or less long chain dicarboxylic acid. In some cases, thesecond phase or light phase is free of long chain dicarboxylic acid.

The fermentation broth can be a sterilized fermentation broth. In someembodiments, a fermentation broth is heated to about 90° C. to about105° C., e.g., about 90° C. to about 105° C., about 90° C. to about 102°C., about 90° C. to about 100° C., about 90° C. to about 98° C., about90° C. to about 96° C., about 90° C. to about 94° C., about 90° C. toabout 92° C., about 91° C. to about 105° C., about 93° C. to about 105°C., about 95° C. to about 105° C., about 97° C. to about 105° C., about99° C. to about 105° C., about 100° C. to about 105° C., about 90° C.,about 91° C., about 92° C., about 93° C., about 94° C., about 95° C.,about 96° C., about 97° C., about 98° C., about 99° C., about 100° C.,about 101° C., about 102° C., about 103° C., about 104° C., or about105° C. prior to adding the acid in step (a) of the method describedherein. In other embodiments, a fermentation broth is heated to about90° C. to about 105° C., e.g., about 90° C. to about 105° C., about 90°C. to about 102° C., about 90° C. to about 100° C., about 90° C. toabout 98° C., about 90° C. to about 96° C., about 90° C. to about 94°C., about 90° C. to about 92° C., about 91° C. to about 105° C., about93° C. to about 105° C., about 95° C. to about 105° C., about 97° C. toabout 105° C., about 99° C. to about 105° C., about 100° C. to about105° C., about 90° C., about 91° C., about 92° C., about 93° C., about94° C., about 95° C., about 96° C., about 97° C., about 98° C., about99° C., about 100° C., about 101° C., about 102° C., about 103° C.,about 104° C., or about 105° C. prior to centrifuging the demulsifiedfermentation broth in step (b) of the method described herein.

In some embodiments, the sterilized fermentation broth is cooled to atemperature ranging from about 80° C. to about 30° C., e.g., about 80°C. to about 30° C., about 75° C. to about 30° C., about 70° C. to about30° C., about 65° C. to about 30° C., about 60° C. to about 30° C.,about 55° C. to about 30° C., about 50° C. to about 30° C., about 45° C.to about 30° C., about 40° C. to about 30° C., about 80° C. to about 40°C., about 80° C. to about 50° C., about 80° C. to about 60° C., about80° C. to about 70° C., about 80° C., about 70° C., about 60° C., about50° C., about 40° C., about 30° C., and the like, prior to heating tothe demulsified fermentation broth in step (a) or step (b) of the methoddescribed herein.

To generate a demulsified fermentation broth having a pH of 6.0 or less,e.g., pH 6.0, pH 5.9, pH 5.8, pH 5.7, pH 5.6, pH 5.5, pH 5.4, pH 5.3, pH5.2, pH 5.1, pH 5.0, pH 4.9, pH 4.8, pH 4.7, pH 4.6, pH 4.5, pH 4.4, pH4.3, pH 4.2, pH 4.1, pH 4.0, pH 3.9, pH 3.8, pH 3.7, pH 3.6, pH 3.5, pH3.4, pH 3.3, pH 3.2, pH 3.1, pH 3.0, pH 2.9, pH 2.8, pH 2.7, pH 2.6, pH2.5, pH 2.4, pH 2.3, pH 2.2, pH 2.1, pH 2.0 or less, an acid can beadded to the fermentation broth. Useful acids include, but are notlimited to, sulfuric acid, hydrochloric acid, nitric acid, acetic acid,phosphoric acid, trifluoromethane sulfonic acid, and any combinationthereof. In some embodiments, the acid is sulfuric acid. In someembodiments, the demulsified fermentation broth has a pH ranging from pH2 to pH 5, e.g., pH 2 to pH 5, pH 2.5 to pH 5, pH 3 to pH 5, pH 3.5 topH 5, pH 4 to pH 5, pH 2.5 to pH 4.5, pH 2.5 to pH 4, pH 2.5 to pH 3.5,and the like. The demulsified fermentation broth has a pH ranging frompH 2.5 to pH 4.

The acid for generating a demulsified fermentation broth can be 2.5M-5Macid, e.g., 2.5M-5M, 2.5M-4M, 3M-5M, 3M-4M, 2.5M-4M, 2.5-3.5M,3.5M-4.5M, 4M-5M, 2.5M-3M, 3M-3.5M, 3.5M-4M, 4M-4.5M, 4.5M-5M, 2.5M, 3M,3.5M, 4M, 4.5M, or 5M, and the like. In some embodiments, 2.5M-5Msulfuric acid, e.g., 2.5M-5M, 2.5M-4M, 3M-5M, 3M-4M, 2.5M-4M, 2.5-3.5M,3.5M-4.5M, 4M-5M, 2.5M-3M, 3M-3.5M, 3.5M-4M, 4M-4.5M, 4.5M-5M, 2.5M, 3M,3.5M, 4M, 4.5M, or 5M hydrochloric acid is added to the fermentationbroth. In certain embodiments, 2.5M-5M sulfuric acid, e.g., 2.5M-5M,2.5M-4M, 3M-5M, 3M-4M, 2.5M-4M, 2.5-3.5M, 3.5M-4.5M, 4M-5M, 2.5M-3M,3M-3.5M, 3.5M-4M, 4M-4.5M, 4.5M-5M, 2.5M, 3M, 3.5M, 4M, 4.5M, or 5Mhydrochloric acid is added to the fermentation broth. In otherembodiments, 2.5M-5M acetic acid, phosphoric acid, nitric acid, ortrifluoromethane sulfonic acid, e.g., 2.5M-5M, 2.5M-4M, 3M-5M, 3M-4M,2.5M-4M, 2.5-3.5M, 3.5M-4.5M, 4M-5M, 2.5M-3M, 3M-3.5M, 3.5M-4M, 4M-4.5M,4.5M-5M, 2.5M, 3M, 3.5M, 4M, 4.5M, or 5M acetic acid, phosphoric acid,nitric acid, or trifluoromethane sulfonic acid is added to thefermentation broth. In some cases, 10%-20% (v/v) sulfuric acid, e.g.,10%-20% (v/v), 11%-20% (v/v), 10%-15% (v/v), 15%-20% (v/v), 11%-15%(v/v), 10% (v/v), 11% (v/v), 12% (v/v), 13% (v/v), 14% (v/v), 15% (v/v),16% (v/v), 17% (v/v), 18% (v/v), 19% (v/v), or 20% (v/v) sulfuric acidis added to the fermentation broth.

In some embodiments, the demulsified fermentation broth has a viscosityof about 5 centipoise (cp) to about 200 cp, e.g., about 5 cp to about200 cp, about 50 cp to about 200 cp, about 100 cp to about 200 cp, about150 cp to about 200 cp, about 5 cp to about 150 cp, about 5 cp to about100 cp, about 5 cp to about 50 cp, about 5 cp, about 10 cp, about 20 cp,about 30 cp, about 40 cp, about 50 cp, about 60 cp, about 70 cp, about80 cp, about 90 cp, about 100 cp, about 110 cp, about 120 cp, about 130cp, about 140 cp, about 150 cp, about 160 cp, about 170 cp, about 180cp, about 190 cp, or about 200 cp. Viscosity can be measured using, forexample, a viscosimeter.

In some embodiments, the first phase (e.g., the heavy phase) and thesecond phase (e.g., the light phase) are produced from the demulsifiedfermentation broth by centrifugation. Centrifugation can be performedvia sequential centrifugation. In some instances, centrifuging isperformed using a scanter centrifuge, such as but not limited to, ahydraulic cyclone centrifuge, a hydrocyclone centrifuge, a horizontalspiral centrifuge, and any combination thereof.

In some embodiments, the long chain dicarboxylic acid is a long chaindicarboxylic acid solid or particle. The long chain dicarboxylic acid ofthe first phase or heavy phase can be isolated or recovered using, forexample, any solid-liquid separation method known by one of ordinaryskill in the art. The isolated long chain dicarboxylic acid can have apurity of at least 95% or higher, e.g., 95%, 96%, 97%, 98%, 99%, 99.5%or more. For instance, the isolated long chain dicarboxylic acid can beabout 97%, 98%, 99%, 99.5% or more pure.

In some embodiments, the isolated long chain dicarboxylic acid isfiltered using, for example, plate frame filtration. In some instances,the isolated long chain dicarboxylic acid is dried. The long chaindicarboxylic acid can be further processed into a crystal form. Theisolated long chain dicarboxylic acid can be filtered, dried, and thenheated to about 90° C. or any other temperature to dissolve thedicarboxylic acid. In some instances, an activated carbon can be addedand maintained at about 90° C. or higher for about 30 minutes to about 2hours. In some cases, the temperature of the dicarboxylic acid mixturecontaining activated carbon is kept at 90° C. for 1 hour. Afterwards,the mixture can be filtered to produce a decolorized solution thatincludes the dicarboxylic acid. The decolorized solution can be cooledto about 28° C. The crystal form of the dicarboxylic acid can beseparated from the decolorized solution by, e.g., centrifugation. Thedicarboxylic acid crystal can be washed, and optionally, dried. Thedried dicarboxylic acid crystal can also be chipped.

In another aspect, described herein is a method for isolating a longchain dicarboxylic acid from a fermentation broth containing microbialcells comprising (a) adding sulfuric acid to the fermentation broth toproduce a demulsified fermentation broth having a pH of 2.5-4; (b)heating the demulsified fermentation broth to a temperature from 90° C.to 100° C.; (c) centrifuging the demulsified fermentation broth togenerate a first phase comprising the long chain dicarboxylic acid and asecond phase comprising the microbial cells, wherein the first phase issubstantially free of the microbial cells and the second phase issubstantially free of the long chain dicarboxylic acid; (d) isolatingthe long chain dicarboxylic acid from the first phase to recover anisolated long chain dicarboxylic acid.

In yet another aspect, described herein is a substantially pure longchain dicarboxylic acid isolated according to any one of the methodsprovided herein. In other words, the long chain dicarboxylic acid isabout 95%, 96%, 97%, 98%, 99% or more pure. In some embodiments, theisolated and substantially pure long chain dicarboxylic acid is 95%,96%, 97%, 98%, 99% or more pure. In other embodiments, the isolateddicarboxylic acid is 100% pure.

The method provided herein can be used to separate, isolate, extract, orrecover long chain dicarboxylic acids from a fermentation broth or otheraqueous mixtures (e.g., fermentation streams). The fermentation brothcan contain one or more microbial cells that can synthesize long chaindicarboxylic acids and nutrients such as sugar to maintain the growth ofthe cells.

In some instances, drying includes dewatering the recovered long chaindicarboxylic acid to reduce the amount or level of water present. Thedried long chain dicarboxylic acid can contain less than 5% water, e.g.,4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%,0.4%, 0.3%, 0.2%, 0.1% or less by w/w or v/v. Water content can bedetermined by using volumetric and coulometric Karl Fischer titrationmethods which are recognized by those of skill in the art.

In some embodiments, the isolated long chain dicarboxylic acid comprisesa solid precipitate or particle of the dicarboxylic acid. In someembodiments, the average size of the particle (e.g., the mean volume ofthe particle) of the long chain dicarboxylic acid ranges from about 0.1μm to about 2000 μm, e.g., about 0.1 μm to about 2000 pm, about 0.1 μmto about 1000 μm, about 0.1 μm to about 500 μm, about 0.1 μm to about100 μm, about 1 μm to about 2000 μm, about 1 μm to about 1000 μm, about1 μm to about 500 μm, about 1 μm to about 100 μm, about 10 μm to about2000 μm, about 10 μm to about 1000 μm, about 10 μm to about 500 μm,about 10 μm to about 400 μm, about 10 μm to about 300 μm, about 10 μm toabout 200 μm, about 10 μm to about 100 μm, about 10 μm to about 500 μm,about 20 μm to about 500 μm, about 30 μm to about 500 μm, about 40 μm toabout 500 μm, about 50 μm to about 500 μm, about 60 μm to about 500 μm,about 70 μm to about 500 μm, about 80 μm to about 500 μm, about 90 μm toabout 500 μm, about 100 μm to about 500 μm, about 10 μm to about 90 μm,about 10 μm to about 80 μm, about 10 μm to about 70 μm, about 10 μm toabout 60 μm, about 10 μm to about 50 μm, about 10 μm to about 40 μm,about 10 μm to about 30 μm, about 20 μm to about 100 μm, about 30 μm toabout 100 μm, about 40 μm to about 100 μm, about 50 μm to about 100 μm,about 60 μm to about 100 μm, about 70 μm to about 100 μm, about 80 μm toabout 100 μm, about 90 μm to about 100 μm, and the like. In certaincases, the average size of the particle is greater than 10 μm, e.g., 11μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 150 μm, 200μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, 500 μm, 550 μm, 600 μm, 650μm, or more.

The substantially pure or pure dicarboxylic acid can be furtherprocessed. In some instances, the dicarboxylic acid is dried. In otherinstances, the substantially pure or pure dicarboxylic acid is filtered,e.g., plate frame filtered. In certain instances, the dicarboxylic acidis filtered, dried, treated with an acid, heated, centrifuged, washed,chipped, or any combination thereof. For instance, the precipitateddicarboxylic acid can be (1) filtered (e.g., plate frame filtered) anddried, (2) treated or contacted with an acid (e.g., acetic acid), (3)heated to about 90° C. to dissolve the dicarboxylic acid, (4) contactedwith an activated carbon, (5) heated to about 90° C. to produce adecolorized solution, and (6) cooled to about 28° C. to formdicarboxylic acid crystals. In some embodiments, the dicarboxylic acidcrystals are washed, dried and chipped.

The long chain dicarboxylic acid of the present disclosure can be asaturated or unsaturated straight chain dicarboxylic acid having 9 to 18carbon atoms, with a carboxyl group at each of the two ends of thechain. In other embodiments, the long chain dicarboxylic acid is asaturated or unsaturated straight chain dicarboxylic acid having 11 to14 carbon atoms, with a carboxyl group at each of the two ends of thechain. The long chain dicarboxylic acid can be selected from the groupconsisting of an undecanedioic acid, dodecanedioic acid, tridecanedioicacid (brassylic acid), tetradecanedioic acid, pentadecanedioic acid,hexadecanedioic acid, and any combination thereof. In some instances,the long chain dicarboxylic acid is an undecanedioic acid, dodecanedioicacid, tridecanedioic acid (brassylic acid), tetradecanedioic acid, orany combination thereof.

IV. EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1. Isolation of Dodecanedioic Acid (DC12) from a FermentationBroth

5 m³ of the fermentation broth of dodecanedioic acid (DC12, produced byShandong Cathay Biomaterials Ltd.) was obtained. The broth contained 12%dodecanedioic acid and 1.2% cells. The fermentation broth was heated to90° C. The heated broth was stirred while a sulfuric acid solution (3mol/L) was added into the fermentation broth. The pH value of themixture was adjusted to 4.5. The temperature was kept at 95° C.-100° C.for 30-60 min during the acidification. The demulsified mixture wasslowly cool down to 60° C., and then centrifuged using, for example, athree-series cyclone system (RWT 1530) at a feed rate of 16 m³/h and afeed pressure of 5-6 kg. Two outflows from the centrifuge were used: onefor the heavy phase which contained most of the dibasic acid particlesand the other for the light phase which contained most of the microbialcells and water. The contents of the light phase were directly flowed towaste water treatment station.

The concentration of all diacids in the fermentation broth was about 90%(according to dry weight analysis) and the concentration of the cellswas about 10% (by dry weight).

The yield of DC12 by the method described herein was 98%. The removalrate of the total cells was 88.9%. The long chain diacid particles werecollected through plate frame filtration. The purity of isolated DC12products was 97.2%, as determined by gas chromatography (GC).

The following method to determine the concentration of cells in thefermentation broth was used in Examples 1 to 5 described herein. About100 g of the sample fermentation broth was weighted to an accuracy oftwo decimal places and recorded as “M1”. A 30% NaOH (% by weight)solution was added to the sample and heated to completely dissolve thelong chain dicarboxylic acids and raise the pH to pH 9.5. The mixturewas cooled down to room temperature, and centrifuged at about 4000 rpmfor about 10 minutes using, for example, a 250 mL centrifuging cup inorder to pellet the microbial cells. The supernatant fluid was decantedand about 100 mL ionized water was added and stirred. The centrifugationstep was repeated, and the supernatant fluid was again decanted. Thepelleted cells were dried at 100° C. to a constant weight. The weightwas recorded of the dried pellet as “M2” (to an accuracy of two decimalplaces). The concentration of the cells in the fermentation broth wascalculated using the formula: C=(M2×100%)/M1.

The total removal rate of the cells was determined by measuring theconcentration of cells of the fermentation broth before treatment (C1)and after isolation of the long chain dicarboxylic acids (C2). The totalremoval rate of cells was calculated using the formula:((C1−C2)×100%)/C1.

The yield of the long chain dicarboxylic acid from the isolation methodwas calculated using the formula: yield=((V2×C2)×100%)/(V1×C1), whereinC1 represents the concentration of long chain dicarboxylic acid in thefermentation broth; V1 represents the volume of fermentation brothtreated using the method described herein; C2 represents theconcentration of long chain dicarboxylic acid in the heavy phase (firstphase), and V2 represents the volume of the heavy phase (first phase).

Example 2. Isolation of Dodecanedioic Acid (DC12) from a FermentationBroth

5 m³ of the fermentation broth of dodecanedioic acid (DC12, produced byShandong Cathay Biomaterials Ltd.) was obtained, of which theconcentration of dodecanedioic acid was 12% and the concentration of thecells was 1.2%. The fermentation broth was stirred while a sulfuric acidsolution (3 mol/L) was added into the broth. The pH value of the mixturewas adjusted to 5.5. Then the mixture was heated to 98° C. andmaintained at this temperature for 20min. The mixture was then slowlycooled down to 35° C. Next, the mixture was centrifuged using, forexample, a horizontal spiral centrifuge (Model 250) at a centrifugalforce of 3000-3100 g and a feed rate of 3.6 m³/h. There were two outflowphases from the centrifuge. One was used for the heavy phase whichcontains most of the dibasic acid particles, and the other was for thelight phase which contains most of the cells and water. The light phasewas directed to a waste water treatment station.

The concentration of all diacid particles in the broth (according to dryweight analysis) was about 90% and the concentration of the cells wasabout 10% (by dry weight).

The yield of DC12 by the method was 98.5%. The total removal rate of thecells was 90.7%. The power consumption of the centrifuge was 50 kwh perton of DC12.

The long chain diacid particles were collected through plate framefiltration. The purity of products was 95% or higher, as determined bygas chromatography (GC). The long chain diacid particles can be useddirectly in the perfume industry. The power consumption of the wholeprocess was 73 kwh per ton of DC12.

Example 3. Isolation of Tetradecanedioic acid (DC14) from a FermentationBroth

5 m³ of the fermentation broth of tetradecanedioic acid (DC14, producedby Shandong Cathay Biomaterials Ltd.) was obtained, of which theconcentration of tetradecanedioic acid was 15% and the concentration ofthe cells was 1.4%. The fermentation broth was stirred as sulfuric acidsolution (3 mol/L) was added into the broth. The pH value of the mixturewas adjusted to 4. The fermentation broth was heated to 100° C. and keptat this temperature for 20 min. The demulsified broth was slowly cooleddown to 30° C. The broth was then centrifuged using a horizontal spiralcentrifuge (Model 250) at a centrifugal force of 2600 g and a feed rateof 5.2 m³/h. Two outflow phases from the centrifuge were used: (1) aheavy phase that included most of dibasic acid particles and (2) a lightphase that included most of cells and water. The low phase was directedto a waste water treatment station.

The concentration of all diacids in the fermentation broth (according todry weight analysis) was about 90% and the concentration of the cellswas about 10% (by dry weight). Using the method described herein theyield of DC14 was 98.2%. The removal rate of the total cells was 90%.

The obtained long chain diacid solution was filtered by plate frame andthen dried to obtain a dry product. The purity for the isolated DC14product was 97.5%, as determined by gas chromatography (GC).

100 kg of the dry long chain diacid product was mixed with 300 kg of 98%acetic acid. The mixture was then heated to 90° C. to dissolve theisolated DC14 product, thereafter activated carbon was added into themixed solution. The mixed solution was kept at 90° C. for 1 hour, andthen filtered to obtain a decolorized DC14 solution. The decolorizedsolution was slowly cooled down to 28° C. The crystalline form wasseparated from the decolorized solution by centrifugation at 1000 rpm.The crystalline DC14 was successively washed using 100 kg of acetic acidat room temperature and 200 kg of pure water, and then centrifuged. TheDC14 product was dried and chipped to obtain a purified, solid DC14product.

Example 4. Isolation of Tridecanedioic Acid (DC13) from a FermentationBroth

5 m³ of the fermentation broth of tridecanedioic acid (DC13, produced byShandong Cathay Biomaterials Ltd.) was obtained. The broth contained 12%tridecanedioic acid and 0.96% cells. The concentration of tridecanedioicacid in the broth was diluted to 9%. The fermentation broth was stirredas sulfuric acid solution (3 mol/L) was added into the broth. As such,the pH of the mixture was adjusted to pH 5.5. The fermentation broth washeated to 95° C. and kept at this temperature for 80 min. Thedemulsified broth was slowly cooled down to 30° C. The resulting brothwas centrifuged using, for example, a horizontal spiral centrifuge(Model 250) at a feed rate of 16 m³/h. Two outflow phases were used fromthe centrifuge: a heavy phase that contained most of the tridecanedioicacid particles and a light phase that contained most of the microbialcells and water. The light phase was directed to a waste water treatmentstation.

The concentration of DC13 in the heavy phase was 95% (according to dryweight analysis). The yield of DC13 by the method described herein was97.1%. The removal rate of the total cells was 88.0%. The powerconsumption of centrifuge was 55 kwh per ton of DC13.

Long chain DC13 particles were recovered after filtration and drying.The purity of the isolated DC13 product was 98%, as determined by gaschromatography (GC). These products can be used directly in fragrancesand the like. The power consumption of the whole process was 70 kwh perton of DC13. The use of centrifugation instead of membrane filtration toseparate the desired long chain dicarboxylic acid provides a higheryield and higher purity compared to conventional or standard methods.

Example 5. Conventional, Prior Art Method for Isolating DodecanedioicAcid (DC12) from a Fermentation Broth

10 m³ of the fermentation broth was obtained. The fermentation brothcontained about 12% DC12, and about 1.6% microbial cells. NaOH solution(30% wt) was added to the fermentation broth to raise the pH to pH 9.5.The mixture was heated to 90° C., and then filtered using a membranefiltration system to remove the cells and obtain a clear broth. Thepower consumption was 320 kwh per ton of DC12.

The clear broth was acidified with a sulfuric acid solution until the pHreached pH 4.5. The acidification process was performed at 95° C.-100°C. for 30-60 min. The resulting mixture was cooled slowly to 60° C., andthen to 25° C.-30° C. quickly. The resulting mixture was filtered usinga plate frame and dried to obtain an isolated DC12 product. The yield ofthe long-chain diacid (the isolated DC12 product) was 97%. The powerconsumption of the whole process was 370 kwh per ton of DC12. Theconsumption of 30% NaOH was 0.04 kg/kg of DC12.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference.

What is claimed is:
 1. A method for isolating a long chain dicarboxylicacid from a fermentation broth containing microbial cells, wherein thelong chain dicarboxylic acid is a saturated or unsaturated straightchain dicarboxylic acid having 9 to 18 carbon atoms, with a carboxylgroup at two ends of the chain, the method comprising: (a) adding anacid to the fermentation broth to produce a demulsified fermentationbroth having a pH of 2-5 or 2.5-4; (b) centrifuging the demulsifiedfermentation broth using a hydraulic cyclone centrifuge, a hydrocyclonecentrifuge, a horizontal spiral centrifuge, or any combination thereofto generate a first phase comprising the long chain dicarboxylic acidand a second phase comprising the microbial cells; and (c) isolating thelong chain dicarboxylic acid from the first phase by filtration torecover an isolated long chain dicarboxylic acid product.
 2. The methodof claim 1, wherein the first phase is substantially free of themicrobial cells.
 3. The method of claim 1, wherein the second phase issubstantially free of the long chain dicarboxylic acid.
 4. The method ofclaim 1, wherein the fermentation broth is a sterilized. fermentationbroth.
 5. The method of claim 1, wherein the fermentation broth is adiluted fermentation broth.
 6. The method of claim 5, wherein thediluted fermentation broth includes at least 0.5%, or about 0.5% toabout 10% microbial cells (dry weight), e.g. 0.5% or 0.6% microbialcells (dry weight).
 7. The method of claim 1, wherein the totalconcentration of dicarboxylic acids in the fermentation broth is atleast about 85% (dry weight), e.g., about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, or more (dry weight).
 8. The method of claim 7, whereinthe total concentration of dicarboxylic acids in the fermentation brothis at least about 90% (dry weight).
 9. The method of claim 1, furthercomprising heating the fermentation broth to a temperature from about90° C. to about 105° C. prior to step (a).
 10. The method of claim 1,further comprising heating the demulsified fermentation broth to atemperature from about 90° C. to about 105° C. prior to step (b). 11.The method of claim 9, further comprising cooling the demulsifiedfermentation broth to a temperature from about 30° C. to about 80° C.after heating.
 12. The method of claim 10, further comprising coolingthe demulsified fermentation broth to a temperature from about 30° C. toabout 80° C. after heating.
 13. The method of claim 1, wherein the acidis selected from the group consisting of sulfuric acid, hydrochloricacid, nitric acid, acetic acid, phosphoric acid, trifluoromethanesulfonic acid, and any combination thereof.
 14. The method of claim 13,wherein the acid is sulfuric acid.
 15. The method of claim 1, whereinthe demulsified fermentation broth has a viscosity of about 5 centipoise(cp) to about 200 cp.
 16. The method of claim 1, wherein centrifugingcomprises sequential centrifugation.
 17. The method of claim 1, furthercomprising drying the isolated long chain dicarboxylic acid.
 18. Themethod of claim
 1. wherein the isolated long chain dicarboxylic acid hasa purity of at least 95% or higher, or at least 97% or higher.
 19. Themethod of claim 1, wherein the long chain dicarboxylic acid is asaturated or unsaturated straight chain dicarboxylic acid having 11 to14 carbon atoms, with a carboxyl group at two ends of the chain.
 20. Themethod of claim 1, wherein the long chain dicarboxylic acid is selectedfrom the group consisting of a nonanedioic acid, decanedioic acid,undecanedioic acid, dodecanedioic acid, tridecanedioic acid,tetradecanedioic acid, pentadecanedioic acid, hexadecanedioic acid,heptadecanedioic acid, octadecanedioic acid, 9-ene-octadecanedioic acid,and any combination thereof, or from the group consisting of anundecanedioic acid, dodecanedioic acid, tridecanedioic acid,tetradecanedioic acid, and any combination thereof.
 21. A method forisolating a long chain dicarboxylic acid from a fermentation brothcontaining microbial cells, wherein the long chain dicarboxylic acid isa saturated or unsaturated straight chain dicarboxylic acid having 9 to18 carbon atoms, with a carboxyl group at two ends of the chain, themethod comprising: (a) adding sulfuric acid to the fermentation broth toproduce a demulsified fermentation broth having a pH of 2.5-4; (b)heating the demulsified fermentation broth to a temperature from 90° C.to 105° C.; (c) centrifuging the demulsified fermentation broth using ahydraulic cyclone centrifuge, a hydrocyclone centrifuge, a horizontalspiral centrifuge, or any combination thereof to generate a first phasecomprising the long chain dicarboxylic acid and a second phasecomprising the microbial cells, wherein the first phase is substantiallyfree of the microbial cells and the second phase is substantially freeof the long chain dicarboxylic acid; and (d) isolating the long chaindicarboxylic acid from the first phase by filtration to recover anisolated long chain dicarboxylic acid.
 22. A method for isolating a longchain dicarboxylic acid from a fermentation broth containing microbialcells, the method comprising: (a) adding an acid to the fermentationbroth to produce a demulsified fermentation broth having a pH of 6 orless; (b) centrifuging the demulsified fermentation broth using ahydraulic cyclone centrifuge, a hydrocyclone centrifuge, a horizontalspiral centrifuge, or any combination thereof to generate a first phasecomprising the long chain dicarboxylic acid and a second phasecomprising the microbial cells; (c) isolating the long chaindicarboxylic acid from the first phase by filtration to recover anisolated long chain dicarboxylic acid, which is dried, and then heatedto dissolve; (d) adding an activated carbon to the dissolveddicarboxylic acid and maintaining the mixture at about 90° C. or higherfor about 30 minutes to about 2 hours, e.g., at 90° C. for 1 hour; (e)filtering the mixture to produce a decolorized. solution that includesthe dicarboxylic acid; (f) cooling the decolorized solution; and (g)separating the dicarboxylic acid in a crystal form from the decolorizedsolution.
 23. The method of claim 22, wherein step (c) comprisestreating or contacting the dried dicarboxylic acid with an acid, e.g.,acetic acid, and heating to about 90° C. to dissolve the dicarboxylicacid, step (f) comprises cooling the decolorized solution to about 28°C. to form dicarboxylic acid crystals, and step (g) further compriseswashing, drying and chipping the dicarboxylic acid crystals.